Rolf Turk PhD, Staff ScientistIntegrated DNA Technologies

Alt-R™ CRISPR-Cas9 System ribonucleoprotein delivery and optimization

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Outline• Ribonucleoprotein complex

– Formation– Stability– Activity

• RNP delivery using lipofection

• RNP delivery using electroporation

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CRISPR genome editing

DNA incorporation or gene knockout• Homology directed repair

vs. non-homologous end joining

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Background information on IDT website

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Implementing CRISPR/Cas9 gene editing

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IDT CRISPR product offerings

3-step transfection using Alt-R™ CRISPR-Cas9 System

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+

+

gRNA complex formation

RNP complex formation

RNP delivery

Step 1

Step 2

Step 3

15 minutes

10–20 minutes

30–60 minutes

T7EI mismatch detection to assay gene disruption

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The Alt-R™ CRISPR-Cas9 System outperforms other methods

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T7 E

ndon

ucle

ase

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avag

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)

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Majority of crRNAs have high editing efficiency

We recommend testing 3 crRNA sites per target gene for a high probability of successful, on-target editing on your first attempt.

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crRNA design

Generating the Cas9 ribonucleoprotein complex

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crRNA

tracrRNA

gRNA complex

crRNA:tracrRNA1:1

RNP:gRNA complex1:1 (lipo)

1:1.2 (electro)Cas9

gRNA complex

RNP

Ribonucleoprotein ease of use—highly stable Cas9 system• Storage of Alt-R™ S.p. Cas9 Nuclease 3NLS

– 60 µM stock (recommend storage at –20C)– Dilute down to 1 µM in:

• Opti-MEM® Media (Thermo Fisher)• Cas9 buffer (20 mM HEPES, 150 mM KCl)• PBS

– Store diluted Cas9 nuclease at 4C for up to 1 month

• Storage of ribonucleoprotein– Can be pre-complexed at 1 µM in:

• Opti-MEM® Media (Thermo Fisher)• Cas9 Buffer (20 mM HEPES, 150 mM KCl)• PBS

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Alt-R S.p. Cas9 Nuclease 3NLS164 kDa60 µM = 60 pmol/µL = 10 µg/µL

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HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8Control -80°C -20°C 4°C

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Cas9 RNP Transfection HEK293, 10 nM RNP, 1.2 µL RNAiMAX®

Cas9 Buffer OptiMEM PBS

HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8

T7 E

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)Cas9 ribonucleoprotein complex stability — 2 weeks

Control

–80˚C –20˚C 4˚C

Delivery of Cas9 ribonucleoprotein

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Lipofection• Mix ribonucleoprotein with

cationic lipids• Incubate (time sensitive)• Apply to cells

http://www.nature.com/scitable/topicpage/what-is-a-cell-14023083

Electroporation• Hard to transfect cells, primary

cells• Mix RNP with cells in

electroporation solution• Electroporate

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HPRT Site 4 HPRT Site 6 HPRT Site 80

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IDT Supplier A Supplier B Supplier C Supplier E Supplier D

T7EI

edi

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efficie

ncy

(%)

HPRT site 4

HPRT site 8

HPRT site 6

T7 E

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Protein comparison—S. pyogenes Cas9 nucleaseCas9 RNP transfection, HEK293, 10 nM RNP, 1.2 µL RNAiMAX®

Gene editing—know your target(s)

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• Targeting locus• Multiple transcript isoforms• SNPs present in your protospacer• Ploidy of cells• Known phenotype?• Will knockout lead to lethality?• Before monoclonal selection, check batch

Cas9 protein is rapidly degraded—less off-target effects

18Liang X, Potter J, et al. (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 208:44–53.

Off-target indel productionWestern blot

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HPRT Site 6 - Cas9 Cells HPRT Site 6 - RNPHPRT Site 8 - Cas9 Cells HPRT Site 8 - RNP

Time post-transfection (hrs.)

T7EI

Cle

avag

e (%

)

HEK293 Cas9 cells: 30 nM, 0.75 µL RNAiMAX®

HEK293 RNP: 10 nM, 1.2 µL RNAiMAX®

On-target editing — time course study

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10 nM RNP150 µL volume= 1.5 pmol (0.25 µg)

T7 E

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avag

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)HPRT Site 6

HPRT Site 8

HPRT Site 6 – Cas9 cells

HPRT Site 8 – Cas9 cells

HPRT Site 6 – RNP

HPRT Site 8 – RNP

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HPRT Site 6 - Cas9 Cells HPRT Site 6 - RNPHPRT Site 8 - Cas9 Cells HPRT Site 8 - RNP

Time post-transfection (hrs.)

T7EI

Cle

avag

e (%

)

HEK293 Cas9 cells: 30 nM, 0.75 µL RNAiMAX®

HEK293 RNP: 10 nM, 1.2 µL RNAiMAX®

On-target editing — time course study

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10 nM RNP150 µL volume= 1.5 pmol (0.25 µg)

48 hrsT7 E

ndon

ucle

ase

I cle

avag

e (%

)HPRT Site 6

HPRT Site 8

HPRT Site 6 – Cas9 cells

HPRT Site 8 – Cas9 cells

HPRT Site 6 – RNP

HPRT Site 8 – RNP

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HEK293-Cas9 Cells HEK293 cells + Ribonucleoprotein Complex

T7 E

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HPRTSite 1

HPRTSite 2

HPRTSite 3

HPRTSite 4

HPRTSite 5

HPRTSite 6

HPRTSite 7

HPRTSite 8

HPRTSite 9

HPRTSite 10

HPRTSite 11

HPRTSite 12

10 nM RNP150 µL volume= 1.5 pmol (0.25 µg)

HEK293 Cas9 Cells: 30 nM, 0.75 µL RNAiMAX®

HEK293 RNP: 10 nM, 1.2 µL RNAiMAX ®

RNP transfection leads to maximal editing

Comparing sources of S. pyogenes Cas9 nuclease

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T7 E

ndon

ucle

ase

I cle

avag

e (%

)

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T7EI

Cle

avag

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)T7

End

onuc

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cleav

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Unmod Alt-R™ crRNA—Cas9 cells

Unmod Alt-R™ crRNA—RNP Modified Alt-R™ crRNA—RNP

CCR5Site 1

CCR5Site 2

CCR5Site 3

CCR5Site 4

CCR5Site 5

CCR5Site 6

CCR5Site 7

CCR5Site 8

CCR5Site 9

CCR5Site 10

CCR5Site 11

CCR5Site 12

HEK293 Cas9 Cells: 30 nM, 0.75 µL RNAiMAX®

HEK293 RNP: 10 nM, 1.2 µL RNAiMAX ®

Modified Alt-R™ crRNA:tracrRNA + Cas9 protein (RNP)

Complete experimental instructions can be found in the User Guide PDF at www.idtdna.com/CRISPR, under the Support tab.

Experimental outline for the Alt-R™ CRISPR-Cas9 System

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Transfection optimization

25RN

P LIPIDSEDITING

TYPE

VIABILITY

CELLS

Transfection optimization

26RN

P

LIPIDS

EDITINGTY

PE

VIABILITY

CELLS

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Toxicity present

Lipofec-tamine® RNAiMAX

Lipid A Lipid B Lipid C Lipid D Lipid E Lipid F Lipid G0

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0.25 µL 0.5 µL 0.75 µL 1.0 µL 1.25 µL 1.5 µL

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Cas9 RNP transfection, HEK293, 10 nM RNP

Lipid optimization—Cas9 RNP transfection

Optimization strategy for lipofection• Follow recommendations from Alt-R™ CRISPR-Cas9 System user

manual• Include positive and negative controls• Pay close attention to cell viability• Use low passage numbers• Cell density should be sub-confluent

• If optimization is necessary:– Test a range of RNP concentrations– Test different amounts of cells per lipofection– Test RNP to cationic lipid ratios

• Test alternatives to lipofection electroporation28

Delivery of RNP by electroporationAmaxa Nucleofector™ (Lonza)• 1-, 16-, or 96-well format• Online database

– Cell lines– Primary cells

• Expensive• ‘Black box’

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Neon™ Transfection System (Thermo Fisher)• Known parameters• Easy optimization• Online database

– Cell lines

• Expensive• Single cuvette• No recommendations

What to look for when doing electroporation • Requires higher ribonucleoprotein concentrations (2–4 µM)• Cas9 ribonucleoprotein complex is not encapsulated

– More accessible to nucleases– More accessible to proteinases

• Electroporation parameters differ per cell line– Amaxa Nucleofector

• Cell database• Optimization protocol (96-well format)

– Neon Transfection System• Optimization protocols

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Optimization strategy for electroporation• Find optimal electroporation parameters• Follow recommendations from IDT User Manual• Include positive and negative controls• Addition of Carrier DNA to boost editing efficiency• Use low passage numbers• Cell density should be sub-confluent

• Pay close attention to cell viability• If necessary, test a range of RNP concentrations• Test different amounts of cells per electroporation

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HPRT Site 6 HPRT Site 8

Ribonucleoprotein complex (µM)

T7EI

Edi

ting

Effici

ency

(%)

3.3 µM RNP30 µL volume= 100 pmol (16 µg)

T7 E

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HPRT Site 6

HPRT Site 8

Nucleofection — Effect of RNP concentration on editing efficiency

Cas9 RNP, HEK293

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0 µM

1 µM

4 µM

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1 µM

4 µM

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1 µM

4 µM

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1 µM

4 µM

10 µ

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No Carrier

20 nt 95 nt 200 nt tRNA

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T7EI

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Effici

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(%) 3.3 µM RNP

30 µL volume= 100 pmol (16 µg)

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)RNP Nucleofection — Effect of carrier DNA properties on editing efficiency

Cas9 RNP, HEK293

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Opt

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µM3.

3 µM

5 µM

10 µ

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Neon Electroporation Nucleofection

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low high

Cell viability

Optimization of Neon System—Alt-R™ CRISPR-Cas9 System RNP

1.5 µM RNP10 µL volume= 18 pmol (3 µg)

T7 E

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Cas9 RNP, HEK293

Advantages of Alt-R™ CRISPR-Cas9 System

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• Guide RNA complex using Alt-R™ CRISPR crRNA and tracrRNA• Quality controlled manufacturing• Ease of use• Modifications for prolonged stability in cellular

environments• Reduced toxicity from innate immune stimulation

• Alt-R™ S.p. Cas9 Nuclease 3NLS• Ease of use• Controlled delivery• Fewer off-target effects• No toxicity• Cost effective

The Alt-R CRISPR Cas9 System

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A complete system• Alt-R™ S.p. Cas9 Nuclease 3NLS• Alt-R™ CRISPR crRNA• Alt-R™ CRISPR tracrRNA• Alt-R™ CRISPR Control Kits (Human, Mouse, Rat)

Delivered as ribonucleoprotein• By cationic lipids:

- Lipofectamine® RNAiMAX® (Thermo Fisher)- Lipofectamine® CRISPRMAX™ (Thermo Fisher)

• By electroporation- Nucleofector™ (Lonza)- Neon™ system (Thermo Fisher)

Visit www.idtdna.com/CRISPR for more information• Support tab

– User guide and short protocols– Previous Alt-R™ CRISPR-Cas9

System webinars– Short tutorial videos on how to

order• Performance tab

– View key data from Alt-R™ CRISPR-Cas9 System experiments

Alt-R™ CRISPR-Cas9 System additional resources

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